Conformation-Selective Analogues of Dasatinib Reveal Insight into Kinase Inhibitor Binding and Selectivity

Abstract

In the kinase field, there are many widely held tenets about conformation-selective inhibitors that have yet to be validated using controlled experiments. We have designed, synthesized, and characterized a series of kinase inhibitor analogues of dasatinib, an FDA-approved kinase inhibitor that binds the active conformation. This inhibitor series includes two Type II inhibitors that bind the DFG-out inactive conformation and two inhibitors that bind the αC-helix-out inactive conformation. Using this series of compounds, we analyze the impact that conformation-selective inhibitors have on target binding and kinome-wide selectivity.

Figure 1.

(A) Overlay of dasatinib (blue) and imatinib (red) kinase inhibitors used in the design of dasatinib DFG-out analogs, DAS-DFGO-I and DAS-DFGO-II. (B) DAS-DFGO-I (white) bound to c-Src (PDB: 4YBJ). P-loop (blue), αC-helix (yellow) and DFG motif (red) are shown. (C) The trifluoromethyl benzamide group is positioned within the DFG pocket, resulting in the DFG-out, inactive kinase conformation.

Figure 2.

(A) Overlay of dasatinib (blue) with the binding space (yellow) of structurally confirmed CHO inhibitors. A para-phenoxy group addition to dasatinib led to the generation of DAS-CHO-I and DAS-CHO-II. (B) DAS-CHO-I bound to c-Src (PDB: 4YBK). (C) An overlay with dasatinib bound to c-Src (PDB: 3G5D) shows the magnitude of the αC-helix shift (~5 Å) and incompatible binding between the CHO inhibitor and the Glu310 side chain of the kinase. (D) DAS-CHO-I bound to c-Abl (PDB: 4YC8). (E) The possible range of αC-helix positioning, from αC-helix in (blue) to c-Abl αC-helix out (green) to c-Src αC-helix out (yellow).

Figure 3.

(A) DAS- and DAS-DFGO-II-BODIPY structures. (B) Determination of BODIPY probes half-life values. A significantly longer half-life measurement was observed for DAS-DFGO-II-BODIPY (red) in comparison to DAS-BODIPY (blue) (C) Kinetic and thermodynamic data comparison for type I and type II BODIPY probes.

Figure 4.

(A) Kinase target comparison for conformation-selective dasatinib analogs utilizing a 50% cutoff value. (B) Analysis of kinase targets amongst the three conformation-selective modes of inhibition. Twenty kinases are targeted equally by the inhibitor set, while six kinases demonstrate a preference for DFG-out binding. Seventeen kinases show shared inhibition between dasatinib and the two DFG-out inhibitors. (C) Kinome tree representation of kinase targets. Colors correspond to the Venn diagram.